Inflammation drives alternative first exon usage to regulate immune genes including a novel iron-regulated isoform of Aim2

  1. Elektra K Robinson
  2. Pratibha Jagannatha
  3. Sergio Covarrubias
  4. Matthew Cattle
  5. Valeriya Smaliy
  6. Rojin Safavi
  7. Barbara Shapleigh
  8. Robin Abu-Shumays
  9. Miten Jain
  10. Suzanne M Cloonan
  11. Mark Akeson
  12. Angela N Brooks
  13. Susan Carpenter

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Abstract

Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2 . We show that this unannotated AFE isoform of Aim2 is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5′UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2 .

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  1. Version published to 10.7554/elife.69431 on eLife
  2. eLife

    ###Reviewer #3:

    In this manuscript, Robinson et al., identified alternative first exon (AFE) switching events conserved between mouse and human following macrophage inflammation. Using short and long-read sequencing, the authors identified a few unannotated transcription initiation sites (TSS) that are specific to an inflammatory response. Among those, they centered on an unannotated TSS in the Aim2 gene that drives expression of a novel isoform regulated by an iron-responsive element in its 5′UTR.

    While previous work had documented crucial AFE switching events in many other biological contexts, Robinson et al. presents here an interesting AFE switching event that can have potential implications for our understanding of the molecular regulation of the innate immune response. I would expect further progress on global mechanisms and biological relevance of these AFE switching events, as well as evidence that the AFE are truly first exons/TSSs.

    Substantive concerns:

    1. Are the AFEs truly first exons/TSS? While both short-read and long-read sequencing detected changes in alternative splicing choices, neither of those are optimal methodologies to analyze first exons. Therefore, I suggest to use a more specialized method to identify (and quantify) more accurately the usage of first exons. Globally, cap analysis of gene expression (CAGE) would be ideal. For validation of specific AFE changes, the qPCR technique has a few issues. First, it does not have nucleotide resolution, so the authors should not refer to TSSs if they used this technique for validation. Second, many downstream first exons are also used as internal exons in other isoforms. There is not a direct technology to analyze specifically first exons/TSSs here. Also, RNA-sequencing technologies, depending on their depth, can definitely miss specific isoforms. Considering a low coverage in 5'end of genes in RNA-seq analysis, this is particularly important for first exons. A qPCR would only analyze the well-known TSSs. Thus, 5'RACE or a similar technology should be performed to assess the relative usage of AFE specifically.

    2. Global mechanism. The authors assumed that the mechanism of AFE switching is generated by transcription initiation and looked for transcription factors binding and chromatin structure modifications in promoters. However, they did not rule out the possibility that the global switching effect is a post-transcriptional regulation, such as differential mRNA stability. A transcription initiation measurement (e.g., 4SU metabolic labelling) is necessary to demonstrate that the changes in AFE usage are co-transcriptional. In addition, in terms of their ATAC-Seq analysis, the chromatin structure changes in promoters can be the cause or consequence of transcription initiation. Thus, it should not be listed as one mechanism driving the expression of AFE events (line 145). Also, to demonstrate a mechanism based on transcription factor binding more than 2 transcription factors should be considered. In any case, the expression patterns of the transcription factors considered are not clear. As a minor note, the bioinformatic analysis of the two promoter regions driving the isoforms of Aim2 (line 156) is not explained in the method section.

    3. Biological relevance. Could the authors evaluate whether the translation regulation of Aim2 based on its AFE switching is a more generalized phenomenon? Are there any global gene regulation changes triggered by the other genes with significant changes in AFE usage?

  3. eLife

    ###Reviewer #2:

    This manuscript by Robinson et al. presents an interesting and timely analysis of a wealth of transcriptome data upon immune stimulation. The unique combination of long-read Oxford Nanopore and short-read Illumina high-throughput sequencing across both human and mouse samples presents an opportunity for many interesting inter-species immune response comparisons, as well as elucidation of full-length transcript information. This paper is well-written and has interesting validation and discussions regarding Aim2. My major concern is that the paper seems to narrow in on the characterization of Aim2 and class of RNA processing changes (alternative first exons) quite quickly without really delving into the rest of the data and how they arrived there. Below are my major/minor comments and suggestions:

    1. I would have liked the authors to provide more insight into how they honed-in on specifically talking about first exon changes, by discussing more of the other RNA processing changes they found. There is cursory mention in the text and figures of other alternative exon or splice site changes. Firstly, other studies (including those referenced by the authors) have found hundreds of RNA processing changes genome-wide upon immune stimulation - especially of cassette exons, alternative splice sites, and last exon/3'UTR changes. However here, the authors only find tens of changes (Fig 1B). Are they underpowered to identify changes and can they do any sort of analyses to show that they are sufficiently powered (# of sequencing reads & junctions, complexity of reads, etc)?

    2. Similarly, I would also be interested in seeing an analysis indicating whether the 50 AFE events that overlap between the long-read and short-read sequencing analyses is a statistically significant overlap. Particularly, how many overlapping events would be expected given the difference in quantification power between the two methods? How many real AFE differences might the authors be missing because the long-read sequencing methods often do not have the power to identify them (ie. lower expressed genes in one or the other condition, thus dropout of isoforms and perhaps fewer isoform differences for differentially expressed genes).

    3. Second, for the non-AFE changes that they did find, there is very little discussion about what those changes might represent. Specifically: (a) how many changes are validated with long-read data?, (b) is there any insight into specific domains being included/changed, especially using the long-read data?, (c) how many of these non-AFE changes overlap between species? and (d) which types of genes show higher overlap between species and what are their characteristics (binding sites, etc)? To my knowledge, this is the first study that is really designed to properly really look at the conservation of splicing or RNA processing changes after immune activation, so I would love to see more analysis and discussion of this aspect genome-wide.

    4. The authors define significant splicing changes as those with a p-value <= 0.25 and |dPSI| >= 10. I'd like some more clarification on whether this is an adjusted p-value (BH, FDR, or some other multiple test-corrected p-value). Especially if this is adjusted, I find it surprising that the authors are choosing such a liberal statistical confidence level and that even with such a liberal threshold, they are only getting tens of significant events. I would like the authors to at least show these same trends across multiple p-value thresholds or with rank threshold analysis (top 5%, top 10%, top 20%) to show biological trends.

    5. The authors introduce their long-read sequencing data by mentioning that they wanted to identify "additional splicing events that are not captured using short-read sequencing." They then go on to only talk about novel first exon events identified with the long-read sequencing data. Did they identify any other non-AFE events in using the long-read that could then be quantified with the short read data? And second, how do they quantify confidence for novel AFE isoforms, when long-read data seems to have lots of issues with properly sequencing the terminal ends of transcripts (particularly the 5' end when polyA primed, as occurs in ONT DirectRNA sequencing)? They mention the use of ATAC-seq data to show putative promoter support, but mention at one point in their methods that ATAC regions within 10kb of AFEs are considered. This seems like it could be a rather large region to be sure that the ATAC peak is specific to a novel AFE - what is the average distance between AFEs? Finally, I would love to also see the incorporation of CAGE-seq data (or other 5'end data) to validate the specific AFEs sites - which I believe the FANTOM consortium has across many human and mouse tissues.

  4. eLife

    ###Reviewer #1:

    Our understanding of the transcriptomic impact of innate immune signaling remains incomplete. Here Robinson et al., use both long and short read RNA sequencing to gain further insight into LPS-induced changes to mRNA isoform expression in human and mouse macrophages. Their studies report the novel observation that the most common change in isoform expression is alternative use of the first exon. Such changes are indicative of transcriptional regulation, and is thus consistent with the known impact of innate immune signaling on activation of multiple transcription factors. Despite some minor concerns with details of the study, as enumerated below, this is a well-executed and important study that will be of interest and importance to many studying innate immunity, as well as those interested in gene regulation.

    Major comments:

    1. In some ways this is minor, but the authors should be careful to not describe alternative first exon use as alternative splicing. While a novel splice junction is created, mechanistically this is driven by changing transcriptional regulation, and then splicing occurs in the only pattern available to that TSS. In general this is described appropriately in the manuscript, but at a few points there is confusing terminology.

    2. An interesting and somewhat surprising point in the manuscript is that 50% of the AFE events don't show an overall change in gene expression. For Aim2, which does change, the authors show that the AFE change is due to activated use of the unannotated TSS in LPS-stimulated cells. For those genes for which AFE use doesn't correlate with a change in gene expression (e.g. Ncoa7, Rcan1, Ampd3 - Fig S3) is there still transcriptional activation of one TSS and transcriptional silencing of the other? In other words, is there coordinated regulation of the two TSSs to ensure overall message abundance doesn't change, or does activation of one TSS inherently shut off the other (more akin to splice site competition in traditional AS)?

    3. The data suggesting that an IRE regulates translation of the induced 5'UTR is compelling, but more work should be done to confirm. Most importantly, the experiment in Figure 4J should be repeated with the deltaIRE version of the unannotated UTR. Also is the IRE regulation controlled upon LPS-stimulation, or just the presence of the IRE element? In other words, what is the distribution of the annotated and unannotated isoforms in the polysome in the absence of LPS (i.e. repeat 4P without LPS)? Can the authors comment on whether the level of iron or the activity of IRP1/2 change in LPS-stimulated cells?

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. Timothy W Nilsen (Case Western Reserve University) served as the Reviewing Editor.

    ###Summary:

    There was significant enthusiasm for the work. However, it seems that considerable effort including additional experiments will be required to firm up the conclusions.

  6. Version published to 10.1101/2020.07.06.190330 on bioRxiv